Biochemistry reactive material and device for eliminating electronegative low-density lipoprotein (ldl) and method for treating blood or plasma ex vivo to eliminate electronegative low-density lipoprotein therein

ABSTRACT

The present disclosure provides a biochemistry reactive material, including a substrate and an enzyme composition immobilized on the substrate. The enzyme composition is selected from a group consisting of a first enzyme, a second enzyme, and a combination thereof. The first enzyme is used for eliminating a glycan residue of an electronegative low-density lipoprotein (electronegative LDL). The second enzyme is used for eliminating ceramide carried by an electronegative low-density lipoprotein. The biochemistry reactive material is capable of eliminating electronegative low-density lipoprotein.

CROSS REFERENCE TO RELATED APPLICATION

The present application is based on, and claims priority from, TaiwanApplication Serial Number 104126050, filed on Aug. 11, 2015, and ChinaApplication Serial Number 201510815421.4, filed on Nov. 23, 2015, thedisclosure of which are hereby incorporated by reference herein in itsentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

A sequence listing submitted as a text file via EFS-Web is incorporatedherein by reference. The text file containing the sequence listing isnamed “0965-A24497-US_Seq_Listing.txt”; its date of creation was Dec.29, 2015; and its size is 11,648 bytes.

TECHNICAL FIELD

The technical field relates to a biochemistry reactive material anddevice for eliminating electronegative low-density lipoprotein (LDL) anda method for treating blood or plasma ex vivo to eliminateelectronegative low-density lipoprotein therein

BACKGROUND

Low-density lipoprotein (LDL) is a kind of lipoprotein that is a productof lipoprotein lipase action. Lipoproteins play a role in lipidtransportation. It has long been known that the level of cholesterolcarried by low-density lipoprotein is associated with the occurrence andpresence of cardiovascular diseases.

At present, in the medical field, plasma LDL cholesterol (LDL-C) isstill used as an indicator for estimating cardiovascular diseases.However, the low-density lipoprotein level in the plasma of patientswith acute myocardial infarction has no tendency to increase.

Due to external factors, such as excess oxidation pressure, etc.,low-density lipoprotein will be post-translation modified, and presentshigher electronegativity to become electronegative LDL or L5.

Electronegative LDL (L5) electronegative low-density lipoprotein is amajor factor for causing cardiovascular disease. L5 is almostundetectable in a normal human body. In addition, it has been in vitroand in vivo verified that L5 will damages vascular endothelial cells andactivate monocytes and platelets, and result in systemic inflammation,atherosclerosis and myocardial infraction.

Therefore, a novel material, device and/or method for eliminating anelectronegative low-density lipoprotein is/are needed.

SUMMARY

The present disclosure provides a biochemistry reactive material,comprising a substrate and an enzyme composition immobilized on thesubstrate. The enzyme composition is selected from a group consisting ofa first enzyme, a second enzyme, and a combination thereof. The firstenzyme eliminates a glycan residue of an electronegative low-densitylipoprotein (LDL). The second enzyme eliminates ceramide carried by anelectronegative LDL. The biochemistry reactive material is capable ofeliminating electronegative low-density lipoprotein.

The present disclosure also provides a biochemistry reactive device,comprising: the biochemistry reactive material as mentioned above, and acontainer for containing the biochemistry reactive material. Thecontainer has at least one inlet and at least one outlet, wherein aliquid sample enters into the biochemistry reactive device from the atleast one inlet, and flows through the biochemistry reactive material toreact with the biochemistry reactive material, and then flows outthrough the at least one outlet.

The present disclosure further provides a method for ex vivo treatingblood or plasma, comprising (a) ex vivo contacting a blood or plasmawith an enzyme composition to react the enzyme composition with theblood or plasma, wherein the enzyme composition is capable ofeliminating low-density lipoprotein. The enzyme composition is selectedfrom a group consisting of a first enzyme, a second enzyme, and acombination thereof. The first enzyme is for eliminating a glycanresidue of an electronegative LDL. The second enzyme is for eliminatingceramide carried by an electronegative low-density lipoprotein. Themethod also comprises (b) terminating the contact between the blood orplasma and the enzyme composition to terminate the reaction of theenzyme composition with the blood or plasma.

A detailed description is given in the following embodiments withreference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

The present invention can be more fully understood by reading thesubsequent detailed description and examples with references made to theaccompanying drawings, wherein:

FIG. 1A is a schematic cross-sectional view of a biochemistry reactivedevice of one embodiment of the present disclosure;

FIG. 1B is a schematic cross-sectional view of a biochemistry reactivedevice of another embodiment of the present disclosure;

FIG. 1C is a schematic cross-sectional view of a biochemistry reactivedevice of another embodiment of the present disclosure;

FIG. 2 shows the transformation result for NEU2;

FIG. 3 shows the transformation result for ASAH2;

FIG. 4 shows the result of gene transfection of NEU4/ASAH2 confirmed bywestern blot;

FIG. 5 shows the result for NEU2 purification;

FIG. 6 shows the result for ASAH2 purification;

FIG. 7 shows apoptosis of endothelial cells of blood vessel co-culturedwith electronegative low-density lipoprotein (electronegative LDL) L5(25 μg/mL; 50 μg/mL) and L5 (1.25 μg) treated by the mmobilized-NEU2filled device for 2 hours (treatment temperature 37° C., pH 7.4) for 24hours, respectively;

FIG. 8 shows results of performing quantitative analysis to the LDLsamples without treatment and treated without enzyme at 37° C. for 2hours or treated with NEU2 for 2 hours (treatment temperature 37° C. pH7.4) to determine the content of L5 therein;

FIGS. 9A, 9B and 9C show that performing a mass spectrometry analysis onL5 can detect L5 specific glycosylation of apoE lipoprotein;

FIGS. 10A-1, 10A-2, 10B-1, and 10B-2 show that the mass spectrometryanalysis result of L5 treated with NEU2, wherein apoE specific glycanresidues have been removed;

FIGS. 11A, 11B-1, 11B-2, 11B-3, and 11B-4 show NEU2, NEU4 immobilized ondifferent material both are capable of effectively eliminatingglycosylation on lipoproteins; Sequences of LDL which are most commonlyglycosylated comprise: 1. (R)IGQDGISTSATTNLK(C) (SEQ ID NO. 3) ofapoB100; 2. (K)VLVDHFGYTK(D) (SEQ ID NO. 4) of apoB100; 3. (K)GVISIPR(L)(SEQ ID NO. 5) of apoB100; 4. (K)SGSSTASWIQNVDTKYQIR(I) (SEQ ID NO. 6)of apoB100; 5. (K)AKPALEDLRQGLLPVLESFK(V) (SEQ ID NO. 7) of apoB100.Furthermore, ITRI-A-01(NEU2), ITRI-CD-01(NEU2), ITRI-Si-Nu-01(NEU4) allare capable of effectively eliminating glycan residues on apoB;

FIG. 12 shows ceramide contents of L5 and L5 treated with ASAH2 for 24hours;

FIG. 13 shows ceramide contents of L5 and L5 treated with ASAH2 for 24hours;

FIG. 14 shows ceramide contents of L5 and L5 treated with ASAH2 in thepresence or absence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25mM CaCl₂) for 2 or 24 hours;

FIG. 15 shows ceramide contents of L5 and L5 treated with ASAH2 in thepresence or absence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25mM CaCl₂) for 24 hours;

FIG. 16A shows the result of quantitative analysis for lipidconstituents of L5 and L5 treated with ASAH2 for 24 hours massspectrometry;

FIG. 16B shows ceramide contents of L5 and L5 treated with ASAH2 in thepresence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl2)for 2 hours;

FIGS. 17A, 17B-1, and 17B-2 show that immobilized ASAH2 is capable ofeffectively eliminating ceramide and increasing a product, sphingosine;One of the most common ceramides of L5 is Cer (d18:0/25:0), and after ithas been catalyzed by ASAH2, a product, sphingosine, is produced. Theexperimental results show that immobilized ASAH2 (ITRI-EC-AS-01) iscapable of reducing Cer (d18:0/25:0) contained by the LDL sample andincreasing the product sphingosine.

DETAILED DESCRIPTION

In the following detailed description, for purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the disclosed embodiments. It will be apparent,however, that one or more embodiments may be practiced without thesespecific details. In other instances, well-known structures and devicesare shown schematically in order to simplify the drawing.

In one embodiment of the present disclosure, the present disclosureprovides a biochemistry reactive material which is capable ofeliminating electronegative low-density lipoprotein (electronegativeLDL). Examples of the electronegative low-density lipoprotein mentionedabove may comprise, but are not limited to, low-density lipoproteins L1,L2, L3, L4, L5, etc. In one embodiment, the electronegative low-densitylipoprotein mentioned above may be low-density lipoprotein L5. Moreover,L5 is the most electronegative and most harmful low-density lipoprotein.

The biochemistry reactive material of the present disclosure maycomprise, but is not limited to, a substrate and an enzyme composition,wherein the enzyme composition is immobilized on the substrate.

Examples of suitable substrate may comprise silica gel, cellulose,diethylaminoethyl cellulose (DEAE cellulose), chitosan, polystyrene,polysulfone, polyethersulfone, acrylate resin, polysaccharide, etc., butthey are not limited thereto. The substrate may have a particlestructure or a hollow-tube structure, etc. In one embodiment, thesubstrate may be a cellulose bead. In another embodiment, the substratemay be a chitosan bead. Moreover, the substrate may be a cellulosehollow fiber, a polysulfone hollow fiber, epoxy acrylic resin or apolyethersulfone hollow fiber, etc.

The preceding enzyme composition may comprise a first enzyme foreliminating a glycan residue of an electronegative LDL, a second enzymefor eliminating ceramide carried by an electronegative LDL, or acombination thereof, but it is not limited thereto. Source organisms ofthe first enzyme and the second enzyme mentioned above have noparticular limitation. In one embodiment, the first enzyme and thesecond enzyme are bioengineered enzymes from human genome and alsopossibly from animal genome.

The preceding first enzyme may be sialidase or glycosidase.

The sialidase may comprise neuraminidase 1 (NEU1), neuraminidase 2(NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and O-sialidasebioengineered from human genome, one of the foregoing enzymes obtainedthrough gene transformation, expression and purification, sialidase froma virus or bacterium (alias, acetylneuraminyl hydrolase), etc., but itis not limited thereto.

Examples of the glycosidase may comprise alpha- and beta-glucosidasebioengineered from human or animal genome, maltase-glucoamylase andsucrase-isomaltase, one of the foregoing enzymes obtained through genetransformation, expression and purification, N-glycosidase F (PNGase F)and glucosidase from a virus, a bacterium or other organism, etc., butthey are not limited thereto.

Furthermore, the second enzyme may be ceramidase.

The ceramidase may comprise N-acylsphingosine amidohydrolase 1 (ASAH1),N-acylsphingosine amidohydrolase 2 (ASAH2), N-acylsphingosineamidohydrolase 2B (ASAH2B), N-acylsphingosine amidohydrolase 2C(ASAH2C), N-acylethanolamine acid amidase, alkaline ceramidase 1,alkaline ceramidase 2, alkaline ceramidase 3, but it is not limitedthereto.

In one embodiment, the enzyme composition in the biochemistry reactivematerial of the present disclosure is the first enzyme. In thisembodiment, the first enzyme mentioned above may be sialidase, but it isnot limited thereto.

In another embodiment, the enzyme composition in the biochemistryreactive material of the present disclosure is the second enzyme. Inthis embodiment, the second enzyme mentioned above may beN-acylsphingosine amidohydrolase 2, but it is not limited thereto.

In another embodiment, the enzyme composition in the biochemistryreactive material of the present disclosure is a combination of thefirst enzyme and the second enzyme. In this embodiment, the first enzymementioned above may be sialidase, but it is not limited thereto, and thesecond enzyme mentioned above may be N-acylsphingosine amidohydrolase 2,but it is not limited thereto.

In another embodiment of the present disclosure, the present disclosureprovides a biochemistry reactive device, and the device can be used foreliminating electronegative low-density lipoprotein in a liquid sample.

Examples of the foregoing liquid sample may comprise an aqueoussolution, a buffer, blood, plasma, etc., but they are not limitedthereto.

Examples of the foregoing electronegative low-density lipoprotein maycomprise electronegative low-density lipoprotein L1, L2, L3, L4 and/orL5, etc., but they are not limited thereto. In one embodiment, theelectronegative low-density lipoprotein mentioned above may beelectronegative low-density lipoprotein L5.

A cross-sectional view of a structure of the biochemistry reactivedevice of the present disclosure is shown in FIG. 1.

Refer to FIG. 1A. The preceding biochemistry reactive device of thepresent disclosure 100 may comprise a biochemistry reactive material 101and a container 103 for containing the biochemistry reactive material101. The container 103 has at least one inlet 105 and at least oneoutlet 107. The foregoing liquid sample enters into the biochemistryreactive device 100 from the inlet 105, and flows through thebiochemistry reactive material 101 to react with the biochemistryreactive material 101, and then flows out through the outlet 107.

The biochemistry reactive material 101 may comprise, but is not limitedto a substrate and an enzyme composition, wherein the enzyme compositionis immobilized on the substrate.

The substrate mentioned above may comprise, but is not limited to,silica gel, cellulose, diethylaminoethyl cellulose, chitosan,polystyrene, polysulfone, polyethersulfone, acrylate resin,polysaccharide, etc. The substrate may have a particle structure or ahollow-tube structure, etc., but it is not limited thereto.

The enzyme composition may comprise, but is not limited to, a firstenzyme for eliminating a glycan residue of an electronegative LDL, asecond enzyme for eliminating ceramide carried by an electronegative LDLor a combination thereof. Source organisms of the first enzyme and thesecond enzyme mentioned above have no particular limitation. In oneembodiment, the first enzyme and the second enzyme are human.

The preceding first enzyme may be sialidase or glycosidase.

The sialidase may comprise, but is not limited to, neuraminidase 1(NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4(NEU4) and 0-sialidase bioengineered from human genome, one of theforegoing enzymes obtained through gene transformation, expression andpurification, sialidase from a virus, a bacterium or other organism,etc.

The glycosidase may comprise, but is not limited to, alpha- andbeta-glucosidase bioengineered from human or animal genome,maltase-glucoamylase and sucrase-isomahase, one of the foregoing enzymesobtained through gene transformation, expression and purification,N-glycosidase F (PNGase F) and glucosidase from a virus, a bacterium orother organism, etc.

In addition, the second enzyme may be ceramidase. The ceramidase maycomprise, but is not limited to, N-acylsphingosine amidohydrolase 1(ASAH1), N-acylsphingosine amidohydrolase 2 (ASAH2), N-acylsphingosineamidohydrolase 2B (ASAH2B), N-acylsphingosine amidohydrolase 2C(ASAH2C), N-acylethanolamine acid amidase, alkaline ceramidase 1,alkaline ceramidase 2, alkaline ceramidase 3.

In one embodiment, the enzyme composition in the biochemistry reactivematerial 101 mentioned above is the first enzyme. In this embodiment,the first enzyme mentioned above may be sialidase, but it is not limitedthereto.

In another embodiment, the enzyme composition in the biochemistryreactive material 101 mentioned above is the second enzyme. In thisembodiment, the second enzyme mentioned above may be N-acylsphingosineamidohydrolase 2, but it is not limited thereto.

In another embodiment, the enzyme composition in the biochemistryreactive material 101 mentioned above is a combination of the firstenzyme and the second enzyme. In this embodiment, the first enzymementioned above may be sialidase, but it is not limited thereto, and thesecond enzyme mentioned above may be N-acylsphingosine amidohydrolase 2,but it is not limited thereto.

Furthermore, a material of the container 103 of the biochemistryreactive device 100 of the present disclosure may comprise glass,acrylic, polypropylene, polyethylene, stainless steel, titanium alloy,etc., but it is not limited thereto. In one embodiment, a material ofthe container 103 of the biochemistry reactive device 100 of the presentdisclosure may be polypropylene. In addition, a shape of the container103 has no particular limitation, and in one embodiment, the container103 is a hollow column.

In one embodiment, as shown in FIG. 1B, the biochemistry reactive device100 of the present disclosure may further comprise a filtering material109 configured in the container 103 behind the at least one inlet 105and at least one outlet 107. Moreover, the pore size of the filteringmaterial mentioned above is smaller than the biochemistry reactivematerial 101 to prevent the biochemistry reactive material 101 leakingfrom the at least one inlet 105 and/or least one outlet 107, but it canallow the liquid sample to pass through. The filtering material 109mentioned above comprises filter paper, glass, acrylic, polypropylene,polyethylene, etc., but it is not limited thereto. In this embodiment,the substrate of the biochemistry reactive material 101 may have aparticle structure or a hollow-tube structure. In one specificembodiment, the substrate of the biochemistry reactive material 101 hasa particle structure, and in this specific embodiment, the substrate ofthe biochemistry reactive material 101 may be a cellulose bead or achitosan bead, but it is not limited thereto.

When the substrate of the biochemistry reactive material 101 is ahollow-tube structure, polyurethane (PU) can be used to package thedevice without using the filtering material 109.

In one embodiment, the container 103 may be a hollow column, and twoends of the hollow column of the container 103 have a first inlet 105 ₁of the inlet mentioned above and a first outlet 107 ₁ of the outletmentioned above, respectively. In this embodiment, the substrate of thebiochemistry reactive material 101 may have a particle structure or ahollow-tube structure.

In another embodiment, as shown in FIG. 1C, the container 103 may be ahollow column, and two ends of the hollow column of the container 103have a first inlet 105 ₁ of the inlet mentioned above and a first outlet107 ₁ of the at least outlet mentioned above, respectively, and a secondinlet 105 ₂ of the inlet and a second outlet 107 ₂ of the outlet arelocated at a side wall of the hollow column. In this embodiment, theliquid sample can enter into the biochemistry reactive device 100 fromthe first inlet 105 ₁ of the container 103, and flows through thebiochemistry reactive material 101, and then flows out through the firstoutlet 107 ₁. Moreover, a second liquid which can be water, a dialysissolution or a salt-containing aqueous solution enters into thebiochemistry reactive device 100 from the first inlet 105 ₂, and flowsthrough the biochemistry reactive material 101, and then flows outthrough the first outlet 107 ₂. The second liquid can bring a by-productout after the reaction or dialysis.

In this embodiment, the substrate of the biochemistry reactive material101 may have a particle structure or a hollow-tube structure. In onespecific embodiment, the substrate of the biochemistry reactive material101 has a hollow-tube structure, and in this specific embodiment, thesubstrate of the biochemistry reactive material 101 may be cellulosehollow fiber, but it is not limited thereto.

In another embodiment of the present disclosure, the present disclosureprovides a method for ex vivo treating blood or plasma. By the methodfor ex vivo treating blood or plasma, an electronegative low-densitylipoprotein in blood or plasma can be eliminated. The foregoingelectronegative low-density lipoprotein may comprise, but is not limitedto, electronegative low-density lipoprotein L1, L2, L3, L4 and/or L5,etc. In one embodiment, the electronegative low-density lipoproteinmentioned above is electronegative low-density lipoprotein L5.

The method for ex vivo treating blood or plasma may comprise thefollowing steps, but it is not limited thereto.

First, a blood or plasma ex vivo contacts with an enzyme composition toreact the enzyme composition with the blood or plasma, wherein theenzyme composition is capable of eliminating electronegative low-densitylipoprotein.

The preceding enzyme composition may comprise a first enzyme foreliminating a glycan residue of an electronegative LDL, a second enzymefor eliminating ceramide carried by an electronegative LDL or acombination thereof, but it is not limited thereto. Source organisms ofthe first enzyme and the second enzyme mentioned above have noparticular limitation. In one embodiment, the first enzyme and thesecond enzyme are human.

The preceding first enzyme may be sialidase or glycosidase.

The sialidase may comprise neuraminidase 1 (NEU1), neuraminidase 2(NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and O-sialidasefrom a human, or one of the foregoing enzymes obtained through genetransformation, expression and purification, sialidase from a virus, abacterium or other organisms, etc., but it is not limited thereto.

Examples of the glycosidase may comprise alpha- and beta-glucosidasebioengineered from human animal genome, maltase-glucoamylase andsucrase-isomaltase, one of the foregoing enzymes obtained through genetransformation, expression and purification, N-glycosidase F (PNGase F)and glucosidase from a virus, a bacterium or other organism, etc., butthey are not limited thereto.

Furthermore, the second enzyme mentioned above may be ceramidase.

The ceramidase may comprise N-acylsphingosine amidohydrolase 1 (ASAH1),N-acylsphingosine amidohydrolase 2 (ASAH2), N-acylsphingosineamidohydrolase 2B (ASAH2B), N-acylsphingosine amidohydrolase 2C(ASAH2C), N-acylethanolamine acid amidase, alkaline ceramidase 1,alkaline ceramidase 2, alkaline ceramidase 3, but it is not limitedthereto.

In one embodiment, the enzyme composition used in the method for ex vivotreating blood or plasma of the present disclosure is the first enzyme.In this embodiment, the first enzyme mentioned above may be sialidase,but it is not limited thereto.

In another embodiment, the enzyme composition used in the method for exvivo treating blood or plasma of the present disclosure is the secondenzyme. In this embodiment, the second enzyme mentioned above may beN-acylsphingosine amidohydrolase 2, but it is not limited thereto.

In another embodiment, the enzyme composition used in the method for exvivo treating blood or plasma of the present disclosure is a combinationof the first enzyme and the second enzyme. In this embodiment, the firstenzyme mentioned above may be sialidase, but it is not limited thereto,and the second enzyme mentioned above may be N-acylsphingosineamidohydrolase 2, but it is not limited thereto.

Furthermore, in one embodiment, the enzyme composition used in themethod for ex vivo treating blood or plasma of the present disclosurecan be immobilized on a substrate. Examples of the substrate maycomprise silica gel, cellulose, diethylaminoethyl cellulose, chitosan,polystyrene, polysulfone, polyethersulfone, resin, polysaccharide, butthey are not limited thereto. Moreover, the substrate may have aparticle structure or a hollow-tube structure.

In the method for ex vivo treating blood or plasma of the presentdisclosure, time for ex vivo contacting the blood or plasma with theenzyme composition may be about 0.25-8 hours. In one embodiment, timefor ex vivo contacting the blood or plasma with the enzyme compositionmay be about 2 hours.

Furthermore, in the method for ex vivo treating blood or plasma of thepresent disclosure, temperature for ex vivo contacting the blood orplasma with the enzyme composition may be about 4-40° C. In oneembodiment, temperature for ex vivo contacting the blood or plasma withthe enzyme composition may be about 37° C.

In addition, in the method for ex vivo treating blood or plasma of thepresent disclosure, the blood or plasma may ex vivo contact with theenzyme composition at about pH 5-10. In one embodiment, the blood orplasma may ex vivo contact with the enzyme composition at about pH 7.4.

Afterward, contact between the blood or plasma and the enzymecomposition is terminated to terminate the reaction of the enzymecomposition with the blood or plasma.

A manner for terminating the contact between the blood or plasma and theenzyme composition has no particular limitation, for example, forterminating the contact between the blood or plasma and the enzymecomposition, the blood or plasma can be separated from the enzymecomposition, or the enzyme composition can be deactivated, etc.

EXAMPLES Example 1 A. Methods

1. Obtainment of Electronegative Low-Density Lipoprotein(Electronegative LDL)

(1) Purifications for Electronegative Low-Density Lipoprotein

Blood samples to be used for LDL isolation were obtained from subjects.After the initial screening, blood samples were removed from thesubjects with precaution against coagulation and ex vivo oxidation. Theplasma was treated with Complete Protease Inhibitor Cocktail (Roche;Cat. No. 05056489001; 1 tablet/100 mL) to prevent protein degradation.

Lipoprotein Preparation from a Human

The plasma was overlaid with 2 mL Milli-Q water and spun at 20,000 rpmfor 2 hours. The upper white fraction and chylomicrons were removed, andthe remnant layer which contains VLDL, IDL, LDL and HDL was saved for aseries of isolation steps.

To progressively separate VLDL (d=0.93-1.006), IDL (d=1.006-1.019), LDL(1.019-1.063 g/dL) and HDL (1.063-1.210 g/dL) from one another, theremnant sample was sequentially adjusted to d=1.006, d=1.019, d=1.063,d=1.210, respectively, by adding potassium bromide, and then the remnantsamples sequentially adjusted to d=1.006, d=1.019 and d=1.063 werecentrifuged at 45,000 rpm for 24 hours at 4° C., and the remnant samplesequentially adjusted to d=1.210 was centrifuged at 45,000 rpm for 48hours at 4° C. After centrifugation at each isolation step, IDL wasdiscarded while VLDL, LDL and HDL were collected. Isolated VLDL, LDL andHDL samples were treated with 5 mM EDTA and nitrogen to avoid ex vivooxidation. After that, VLDL, LDL and HDL samples were dialyzed againstbuffer A (20 M, pH 8.0, 0.5 M EDTA) for 24 hours (×3 times) to removeexcessive potassium bromide, and were filtrated through 0.22-μm filter(Sartorius; Minisart®) to sterilize the samples.

(2) LDL Subfractions

Approximately 30 mg of LDL material was injected onto a UnoQ12anion-exchange column (BioRad) by using the ÄKTA fast-protein liquidchromatography (FPLC) pump (GE Healthcare Life Sciences, Pittsburgh,Pa.). LDL was eluted according to electronegativity by the use of amultistep gradient of buffer B (1 mol/L NaCl in buffer A) at a flow rateof 2 mL/minute. In short, samples were equilibrated with buffer A for 10minutes, followed by being linearly increased to 15% buffer B in 10minutes (fraction 1), linearly increased to 20% buffer B in 30 minutes(fraction 2, 3), kept at 20% buffer B for 10 minutes (fraction 4) andlinearly increased to 100% buffer B in 20 minutes (fraction 5). Lastly,the effluents were monitored at 280 nm.

(3) Purification of Fractionated LDL

Based on the gradient profile, each of the LDL fractions were pooled.The volume of each subfraction was constant. Dilution of LDL duringchromatography depended on the injection volume. The respectivefractions were concentrated with Centriprep® filters (YM-30; EMDMillipore Corp., Billerica, Mass.), dialyzed against buffer A (20 M,pH8.0, 0.5 M EDTA) for 24 hours (3 days) and sterilized by passingthrough 0.22-μm filters (Sartorius; Minisart®). The isolated fractionswere quantified at their protein concentrations by the Lowry method andthen stored at 4° C.

2. Screening of NEU2 or NEU4

(1) Transformation (Gene Cloning for pCMV6 Vector with NEU2 and NEU4Genes)

NEU2 (neuraminidase 2) and NEU4 (neuraminidase 4) were purchased fromOrigene, RC219858 and RC203948. Genes were amplified by ECOS™ 101 DH5αCompetent Cells (Yeastern, FYE608) according to the manufacturer'sdirections.

In short, 1 vial of competent cells with 5 μL plasmid was vortexed for 1second and then incubated on ice for 5 minutes. After 45 secondheat-shock at 42° C., the mixture was plate on LB agar with Kanamycin.

Colonies were checked with PCR by VP1.5 and XL39 primers. Procedures ofthe PCR comprises: 95° C. for 1 minute for pre-PCR denaturation; 2cycles of 95° C. for 10 seconds, 62° C. for 20 seconds, 72° C. for 4minutes; 2 cycles of 95° C. for 10 seconds, 60° C. for 20 seconds, 72°C. for 4 minutes; 2 cycles of 95° C. for 10 seconds, 58° C. for 20seconds, 72° C. for 4 minutes; 15 cycles of 95° C. for 10 seconds, 56°C. for 20 seconds, 72° C. for 4 minutes; 72° C. for 10 minutes forpost-PCR incubation and holding on 4° C.

(2) Plasmid Extraction

After confirming the insertion of transformed colonies, transformedcells were plate-out into 5 ml LB broth with 25 mg/ml kanamycin, andthen incubated at 37° C. overnight.

Plasmid DNA was extracted according to the protocol of Plasmid MiniprepPlus Purification Kit (GeneMark, DP01P). In short, the bacteria werecentrifuged for 1 minute at 14,000×g, and the media was removed. Thepellet was re-suspended in 200 μL Solution I by pipetting, then 200 μLSolution II was added therein and mixed by inverting the tube. 200 μLSolution III was added to the tube and mixed by inverting the tube 5times. The lysate was centrifuged at top speed for 5 minutes and acompact white pellet formed along the side of the tube. The spin columnwas inserted into a collection tube, and the clear lysate was moved tospin column and spun at top speed for 1 minute. The flow-through wasdiscarded, and 500 μL Endotoxin Removal Wash Solution was loaded to thespin column and kept for 2 minutes to equilibrate the membrane, thenspun at top speed for 1 minute. The filtrate was discarded, and 700 μLWashing Solution was added to the spin column and spun at top speed for1 minute, and then this step was repeated. The filtrate was discardedand the spin column was centrifuged for 5 minutes at top speed to removeresidual traces of ethanol. The spin column was transferred into a newtube and 35 μL H₂O was added to the spin column and kept for 1-2 minutesand the tube was centrifuged at top speed for 2 minutes to elute theDNA. The DNA quantified by microplate spectrophotometer (Epoch, BioTek).

(3) Transfection on HEK Cells and Protein Purification

One day before transfection, 1.25*10⁵ HEK293T cells were placed in 500μL DMEM medium in 24-well plate. For each well of cells to betransfected, 1 μg of DNA was diluted in 100 μL serum-free medium, and1.5 μL of Lipofectamine 2000 Transfection Reagent (Invitrogen) was addthereto and mixed gently and incubated for 30 minutes at roomtemperature. After incubation, the complex was added to each wellcontaining cells and mixed gently. The cells were incubated at 37° C. ina CO₂ incubator for 20 hours. The transfected cells were lysed by RIPAwhich containing protease inhibitor to prepare to purify the proteins.

In short, 80 μL ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich) wereequilibrated for one-well cell lysate purification. After protein-resinbinding at 4° C. overnight, the bound FLAG fusion protein was eluted bycompetitive elution with 150 μg/ml 3× FLAG peptide for 2 times, theeluate was collected, and the protein checked by western blot.

3. Efficacy Test for NEU2 or NEU4

(1) Protein Quantification

Pierce BCA Protein Assay Kit (Thermo) was used for proteinquantification according to the manufacturer's directions.

In short, 25 μL serial diluted BSA standard and 5 μL sample in 20 μLsample diluent were pipetted into a 96-well microplate. To prepare BCAworking reagent, 50 parts of BCA Reagent A was mixed with 1 part of BCAReagent B and placed on ice until use. 200 μL of the BCA working reagentwas added to each well and mixed thoroughly, and the plate was coveredand incubated at 37° C. for 30 minutes. The absorbance at 562 nm wasmeasured by spectrophotometer (Epoch, BioTek).

(2) Apoptosis Measurements

Endothelial cells were used after 3 or 4 passages and maintained in DMEM(Invitrogen™, Thermo Fisher Scientific) containing 10% FBS. Duringtreatment, FBS was reduced to 5% in DMEM. 1×10⁴ cells were seeded in96-well plate for 24 hours for subconfluent cultures, and the culturedcells were exposed to PBS (lipoprotein-free, negative control) or graded(25, 50, and 100 μg/mL) LDL subfractions, unfractionated normolipidemicLDL, and LDL/L1/L5 incubated with sialidase for 24 hours. Apoptosis wasassessed with visualization by a Zeiss Axiovert 200 fluorescencemicroscope and filters to capture digital images based on Hoechst 33342,propidium iodide (red), and calcein AM (green) staining of nuclear,apoptotic DNA membrane integrity and cytoplasm respectively according tothe protocol of the manufacturer (Invitrogen™, Thermo FisherScientific).

(3) LC/MS^(E) Analysis for Protein Composition

LDL subfractions were quantified the protein contents by use ofquantitative proteomics techniques utilizing serially coupled liquidchromatography data-independent parallel-fragmentation mass spectrometry(LC/MS^(E)). Such analysis has been shown to be highly quantitative withrespect to both relative and/or absolute (when incorporating spikedinternal peptide standards in the data collection/analysis procedures)protein abundance in complex protein mixtures. Quantitative analysis wasperformed essentially as previously described (PMCID: PMC3816395; PureAppl Chem. 2011; 83(9): 10.1351/PAC-CON-10-12-07. Chemicalcomposition—oriented receptor selectivity of L5, a naturally occurringatherogenic low-density lipoprotein), except on a Waters nanoACQUITYUPLC System and Xevo® G2-XS QTof mass spectrometer (Waters Corporation,MA, USA).

In brief, total proteins isolated from each LDL subfraction were firstdigested with trypsin, and the resulting tryptic peptides werechromatographically separated on a Nano-Acquity separations module(Waters Corporation, MA, USA) incorporating a 50 fmol-on-column trypticdigest of yeast alcohol dehydrogenase as the internally spiked proteinquantification standard. Peptide elution will be executed through a 75im×25 cm BEH C-18 column under gradient conditions at a flow rate of 300nL/minute over 30 minutes at 35° C. The mobile phase was composed ofacetonitrile as the organic modifier and formic acid (0.1% v/v) formolecule protonation. Mass spectrometry was performed on a Xevo® G2-XSQTof instrument equipped with a nano-electrospray ionization interfaceand operated in the data-independent collection mode (MSE). Parallel ionfragmentation was programmed to switch between low (4 eV) and high(15-45 eV) energies in the collision cell, and data was collected from50 to 2000 m/z utilizing glu-fibrinopeptide B as the separate datachannel lock mass calibrant. Data was processed with ProteinLynxGlobalServer v2.4 (Waters). Deisotoped results were searched for proteinassociation from the Uniprot (www.uniprot.org) human protein database.

4. Screening of ASAH2

(1) Transformation (Gene Cloning for pCMV6 Vector with ASAH2 Genes):

ASAH2 (N-acylsphingosine amidohydrolase 2) was purchased from Origene,RC203706. Genes were amplified by ECOS™ 101 DH5α Competent Cells(Yeastern, FYE608) according to the manufacturer's directions.

In short, 1 vial of competent cells with 5 μL plasmid was vortexed for 1second and then incubated on ice for 5 minutes. After 45 secondheat-shock at 42° C., the mixture was plate on LB agar with Kanamycin.

Colonies were checked with PCR by VP1.5 and XL39 primers. Procedures ofthe PCR comprise: 95° C. for 1 minute for pre-PCR denaturation; 2 cyclesof 95° C. for 10 seconds, 62° C. for 20 seconds, 72° C. for 4 minutes; 2cycles of 95° C. for 10 seconds, 60° C. for 20 seconds, 72° C. for 4minutes; 2 cycles of 95° C. for 10 seconds, 58° C. for 20 seconds, 72°C. for 4 minutes; 15 cycles of 95° C. for 10 seconds, 56° C. for 20seconds, 72° C. for 4 minutes; 72° C. for 10 minutes for post-PCRincubation and holding on 4° C.

(2) Plasmid Extraction

After confirming the insertion of transformed colonies, transformedcells were plate-out into 5 ml LB broth with 25 mg/ml kanamycin, andthen incubated at 37° C. overnight.

Plasmid DNA was extracted according to the protocol of Plasmid MiniprepPlus Purification Kit (GeneMark, DP01P). In short, the bacteria werecentrifuged for 1 minute at 14,000× g, and the media was removed. Thepellet was re-suspended in 200 μL Solution I by pipetting, then 200 μLSolution II was added therein and mixed by inverting the tube. 200 μLSolution III was added to the tube and mixed by inverting the tube 5times. The lysate was centrifuged at top speed for 5 minutes and acompact white pellet formed along the side of the tube. The spin columnwas inserted into a collection tube, and the clear lysate was removed tospin column and spun at top speed for 1 minute. The flow-through wasdiscarded, and 500 μL Endotoxin Removal Wash Solution was loaded to thespin column and kept for 2 minutes to equilibrate the membrane, thenspun at top speed for 1 minute. The filtrate was discarded, and 700 μLWashing Solution was added to the spin column and spun at top speed for1 minute, and then this step was repeated. The filtrate was discardedand the spin column was centrifuged for 5 minutes at top speed to removeresidual traces of ethanol. The spin column was transferred into a newtube and 35 μL H2O was added to the spin column and kept for 1-2 minutesand the tube was centrifuged at top speed for 2 minutes to elute theDNA. The DNA quantified by microplate spectrophotometer (Epoch, BioTek).

(3) Transfection on HEK Cells and Protein Purification

One day before transfection, 1.25*10⁵ HEK293T cells were placed in 500μL DMEM medium in 24-well plate. For each well of cells to betransfected, 1 μg of DNA was diluted in 100 μL serum-free medium, and1.5 μl, of Lipofectamine 2000 Transfection Reagent (Invitrogen) was addthereto and mixed gently and incubated for 30 minutes at roomtemperature. After incubation, the complex was added to each wellcontaining cells and mixed gently. The cells were incubated at 37° C. ina CO₂ incubator for 20 hours. The transfected cells were lysed by RIPAwhich containing protease inhibitor to prepare to purify the proteins.

In short, 80 μL ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich) wereequilibrated for one-well cell lysate purification.

After protein-resin binding at 4° C. overnight, the bound FLAG fusionprotein was eluted by competitive elution with 150 μg/ml 3× FLAG peptidefor 2 times, the eluate was collected, and the protein checked bywestern blot.

5. Efficacy Test for ASAH2

(1) Protein Quantification

Pierce BCA Protein Assay Kit (Thermo) was used for proteinquantification according to the manufacturer's directions.

In short, 25 μL serial diluted BSA standard and 5 μL sample in 20 μLsample diluent were pipetted into a 96-well microplate. To prepare BCAworking reagent, 50 parts of BCA Reagent A was mixed with 1 part of BCAReagent B and placed on ice until use. 200 μL of the BCA working reagentwas added to each well and mixed thoroughly, and the plate was coveredand incubated at 37° C. for 30 minutes. The absorbance at 562 nm wasmeasured by spectrophotometer (Epoch, BioTek).

(2) Lipid Extraction

30 μg LDL/L1/L5 were incubated with 5 μg ASAH2 in ASAH2 buffer (200 mMTris-HCl at pH 8.4, 1.5 M NaCl, 25 mM CaCl₂) at 37° C. After 2 or 24hours incubation, samples were transferred to a glass tube. 1 mL H₂O,2.5 mL methanol and 1.25 mL CHCl₃ were added to samples, and vortexedfor 15 seconds. Then, additional 0.9 mL H₂O and 1.25 mL CHCl₃ wereapplied to samples, vortexed for 15 seconds, and centrifuged at 3000 rpmfor 10 minutes. Bottom layer organic solvents were transferred to a 2.0mL glass tube using a glass syringe. Each sample was flushed withnitrogen until dry pallets, and dissolved with 0.25 mL sample solution(isopropanol/acetonitrile/H₂O=2:1:1).

(3) LC/MS^(E) Analysis for Lipid Composition

Total lipids, phospholipids, neutral lipids and free fatty acid fromeach subfractions of LDL were quantified the lipid contents by use ofliquid chromatography data-independent parallel-fragmentation massspectrometry (LC/MS^(E)). Quantitative analysis was performedessentially as previously described.

In brief, lipids were chromatographically separated on a ACQUITY UPLCSystem (Waters Corporation, MA, USA) incorporating a CSH™ 1.7 μm, 2.1mm×10 cm C-18 column under gradient conditions at a flow rate of 400μL/minute over 18 minutes at 55° C. The mobile phase A will be composedof 10 mM NH4HCO₂ in ACN/H₂O (60/40) and 0.1% formic acid (0.1% v/v),mobile phase B will be composed of 10 mM NH₄HCO₂ in IPA/ACN (90/10) and0.1% formic acid (0.1% v/v) for molecule protonation. Mass spectrometrywas performed on a Xevo® G2-XS QT of instrument equipped with anelectrospray ionization interface and operated in the data-independentcollection mode (MSE). Parallel ion fragmentation was programmed toswitch between low (4 eV) and high (35-55 eV) energies in the collisioncell, and data was collected from 50 to 1600 m/z utilizing leucin as theseparate data channel lock mass calibrant. Data was processed withMarkerLynx (Waters).

B. Results

1. Transformation

(1) NEU2

Transformation result for NEU2 is shown in FIG. 2.

According to FIG. 2, it is known that NEU2 transformations for colonies3, 5 and 6 (see lane 3, 5 and 6, respectively) were successful.Therefore, colonies 3, 5 and 6 were selected to be amplified, andplasmid of NEU2 was stocked.

(2) ASAH2

Transformation result for ASAH2 is shown in FIG. 3.

Colony 7 was selected to be amplified, and plasmid of ASAH2 was stocked.

2. Transfection

Transfection of NEU4/ASAH2 genes was confirmed by western blot, and theresult is shown in FIG. 4.

Conditions for the gene transfection are shown in the following:

HEK293T 1.25×10⁵ cells in 24 well

Plasmid: NEU4 and ASAH2

DNA amount: 1 μg

Transfected by Lipofectamine

SDS-PAGE: using 5 μl sample

Primary antibody: anti-DDK (1:2000)

3. Protein Purification

(1) NEU2 Purification

The result for NEU2 purification is shown in FIG. 5. FIG. 5 shows thatNEU2 was indeed purified. The amino acid sequence of NEU2 is shown asSEQ ID NO. 1.

(2) ASAH2 Purification

The result for ASAH2 purification is shown in FIG. 6. FIG. 6 shows thatASAH2 was indeed purified (extract 1 and extract 2 are proteins obtainedfrom different batches). The amino acid sequence of ASAH2 is shown asSEQ ID NO. 2.

Example 2 Enzyme Immobilization

Method 1

0.4454 g heat-activated silica gel was placed in 7 mL CHCl₃, and APTSwas added therein by a weight of 1/5 weight of heat-activated silica gelto form a mixture. After stirring at room temperature for 24 hours, themixture was filtered. The obtained solid was drained in vacuum at 50° C.After the solid was drained, 5% glutaraldehyde (phosphate Buffer, pH=8,IX TBS) was added to the solid, and stirred for 21 hours to form asolution. The solid in the solution was filtered out and washed withwater and a solid substance was obtained. NEU2 (1/100-10000 wt %) wasadded to the solid substance and diluted with phosphate buffer, 1×TBSpH=8 to a volume of 2 mL, and reacted with the solid substance at roomtemperature for 24 hours. Finally, the solid substance was filtered outand washed with phosphate buffer (pH=8) and an enzyme immobilizedproduct (ITRI-Siw-Nu-01) was obtained.

Method 2

3-glycidoxypropyltrimethoxysilane was added to heat-activated silica gelin toluene by a weight of 1/5 weight of heat-activated silica gel,refluxed for 20 hours, and then filtered. The obtained solid was washedwith acetone and then drained in vacuum. NEU2 (1/100-10000 wt %) wasadded to the solid and stirred in phosphate buffer for 2 hours and 15minutes, and then the solid was filtered out. The solid was washed withdeionized water and a buffer (pH 8) to obtain an enzyme immobilizedproduct.

Method 3

1 g cellulose beads in 15 mL water were adjusted to about pH 11 by aNaOH solution, and then 1 g cyanogen bromide was added therein at roomtemperature. After about 30 minutes, the cellulose beads were washed indeionized water and a phosphate buffer (pH 8) in order. NEU2 in aphosphate buffer was added to the cellulose beads by a weight ratio of1/600, and stirred overnight. After that, the cellulose beads werewashed in a phosphate buffer (pH 8) to obtain an enzyme immobilizedproduct.

Method 4

0.5 g cellulose beads in 1.5 mL water were refluxed in 10 mL toluene,and then cellulose beads were filtered out and washed with acetone and aphosphate buffer (pH 8). After that, the cellulose beads were added to5% (w/v) glutaraldehyde (phosphate buffer, pH 8) and stirred at roomtemperature for 21 hours. Afterward, the cellulose beads were filteredout, and washed in a phosphate buffer (pH 8) to obtainglutaraldehyde-activated-cellulose beads. NEU2 in a phosphate buffer wasadded to the cellulose beads by a weight ratio of 2/1000, and stirredovernight. After that, the cellulose beads were washed in a phosphatebuffer (pH 8) to obtain an enzyme immobilized product.

Method 5

Hypogel® 200NH₂ were added to 5% (w/v) glutaraldehyde (phosphate buffer,pH 8) and stirred at room temperature for 21 hours. Afterward, the solidsubstance was filtered out, and washed with a phosphate buffer (pH 8) toobtain glutaraldehyde-activated gel. NEU2 (1/10000 wt %) was dilutedwith a phosphate buffer (pH=8) to a volume of 15 mL, and mixed with 1.13g of the glutaraldehyde-activated gel at room temperature for 20 hours.Finally, the solid was filtered out and washed in a phosphate buffer(pH=8) to obtain an enzyme immobilized product.

Method 6

1 g diethylaminoethyl cellulose (DEAE cellulose) was washed in water,suspended in an NaOH solution (1 M aqueous solution), stirred for 10minutes, and then filtered out and washed in water. The obtained solidsubstance was suspended in 10 mL dioxane to form a suspension. 2 gcyanuric chloride and 10 mL toluene were added to the suspension andstirred for 30 minutes and then the solid therein was filtered out. Thesolid was washed with dioxane, water and acetone in order and driedunder reduced pressure to form an activated solid support. After that,NEU2 1/10000 (wt %) was added to the activated solid support and stirredfor 18 hours. Afterward, the activated solid support was filtered outand washed in water to obtain an enzyme immobilized product.

Method 7

0.5 g chitosan beads were added to 10 mL 0.5% glutaraldehyde, andstirred at room temperature for 1 hour, and then washed with water,continuously and thoroughly to form activated beads. After that, theactivated beads were reacted with NEU2 1/3500 (wt %) at room temperaturefor 2 hours, filtered out and then washed with deionized water to obtainan enzyme immobilized product.

Method 8

1 g cellulose hollow fiber, as per the procedures in Method 4, wasactivated by APTS and glutaraldehyde, and then reacted with NEU2 3/10000(wt %) in phosphate buffer (pH=8), stirred overnight, and washed with aphosphate buffer (pH=8) to obtain an enzyme immobilized product.

Method 9

1 g cellulose hollow fiber, as per the procedures in Method 3, wasactivated by cyanogen bromide, and then reacted with NEU2 in phosphatebuffer (pH=8), stirred overnight, washed with a phosphate buffer (pH=8)to obtain an enzyme immobilized product.

Method 10

ECR-8204F epoxy-acrylate resin was washed in deionized water, reactedwith ASAH2 1/10000 (wt %), adjusted to a volume of 2 mL with a 0.2 Msodium phosphate buffer, and then stirred for 24 hours. After that,epoxy-acrylate resin was filtered out and washed in deionized water and2M phosphate buffer (pH=8) to obtain about 52 mg of enzyme immobilizedproduct (ITRI-EC-AS-01).

Method 11

Iontosorb MT200 cellulose beads were washed in deionized water. Next,the cellulose beads were washed with 3:7 water/dioxane, 7:3water/dioxane, 100% dioxane in order. After that, dioxane was added tothe cellulose beads, and CDI was added therein by a weight of 1/3 weightof cellulose beads, and stirred for about 0.5-1 hour to form a solution.The dioxane in the solution was removed under reduced pressure, and thenNEU 2 was immediately added therein and stirred for about 2 hours and 15minutes. After the reaction, the cellulose beads in the solution werefiltered out and were washed in a buffer (pH=6.5) to obtain a wetproduct about 0.2 g (ITRI-CD-01).

Method 12

0.5 μg NEU2 was added to 2% w/v alginate aqueous solution to form amixture solution. Next, the mixture solution was dropped into a stirring2% CaCl₂ (w/v) aqueous solution by a syringe needle. After that, theCaCl₂ aqueous solution was continuously stirred for 30 minutes, and thenparticles formed in the CaCl₂ aqueous solution were filtered out andwashed in deionized water to obtain a wet product (ITRI-A-01).

Example 3 Efficacy of Immobilized-NEU2 Filled Device

NEU2 was immobilized by Method 2 in Example 2, and then the immobilizedNEU2 was filled into a tube to form a biochemistry reactive device(immobilized-NEU2 filled device) shown in FIG. 1B.

(1) Determination of Apoptosis

Endothelial cells of blood vessel were co-cultured with electronegativelow-density lipoprotein (electronegative LDL) L5 (25 μg/mL; 50 μg/mL)and L5 (1.25 μg) which was treated by the mmobilized-NEU2 filled devicefor 2 hours (treatment temperature 37° C., pH 7.4) for 24 hours,respectively. After that, apoptosis of the endothelial cells wasdetermined, and the results are shown in FIG. 7.

According to FIG. 7, it is known that 25 μg/mL L5 results in apoptosisto about 15% endothelial cells and 50 μg/mL L5 results in apoptosis toabout 30% endothelial cells while after the treatment of 1.25 μg NEU2,apoptosis effect of L5 to endothelial cells is reduced.

(2) Quantitative Analysis for Electronegative Low-Density Lipoprotein(Electronegative LDL)

LDL samples were obtained from a heart disease patient. Quantitativeanalysis for L5 was performed on the LDL samples without treatment andthose treated without enzyme at 37° C. for 2 hours or treated with NEU2for 2 hours (treatment temperature 37° C. pH 7.4) to determine thecontent of L5 in the samples mentioned above. The results are shown inFIG. 8.

According to FIG. 8, it is known that after being treated with NEU2enzyme for 2 hours, L5 content of the LDL sample was decreased from12.4% to 8.48%.

(3) Mass Spectrometry

Mass spectrometry analysis was performed on L5 and L5 treated with NEU2for 2 hours (treatment temperature 37° C., pH 7.4). The results areshown in FIGS. 9A, 9B and 9C.

It has been known that the feature of L5 is that serine and threonine ofapolipoprotein E (apoE) are usually glycosylated.

Refer to FIGS. 9A and 9B. Molecular weight 1497 indicates non-toxic LDL.Molecular weight of LDL with one glycosyl molecule is 1700, molecularweight of LDL with two glycosyl molecules is 1884, and molecular weightof LDL with three glycosyl molecules is 2154. FIG. 9C shows that theamino acid sequence of apolipoprotein E is glycosylated, and thatresults in the charge-to-mass ratio of the original peptide chain beingincreased from 1497.8009 to 1700.8868, 1884.9021 and 2154.0300.

FIGS. 10A1-2 show that there is no molecule with a charge-to-mass ratioof 1700, 1884 or 2154 that is detected for L5 treated by theimmobilized-NEU2 filled device for 2 hours, and that indicates thatthere is no glycosylation on serine and threonine of apolipoprotein E,i.e., the glycan residues of LDL have been removed.

Similarly, FIGS. 10B1-2 show that for L5 treated by the immobilized-NEU2filled device for 2 hours, there is no glycosylation on other sites ofapolipoprotein E, and that indicates that the glycan residues of LDLhave been removed.

Example 4 Efficacy of ASAH2

(1) LC/MS^(E) Analysis for L5 Treated with ASAH2 for 24 Hours

L5 was treated with ASAH2 for 24 hours. LC/MS^(E) analysis was performedon L5 without treatment and L5 with the preceding treatment to determinethe ceramide content in the L5 samples mentioned above (for the detailedexperimental methods, please see “5. Efficacy test for ASAH2” in “A.Method” of Example 1).

The results for LC/MS^(E) analysis are shown in Table 1 (the four valuesshown in each group were obtained from determining the same sample fourtimes). Conversion was performed to signal of each sample in Table 1 toobtain ceramide content percentage of each sample (the highest signal ofthe L5 without treatment was set as 100%), and the results are shown inFIG. 12. ASAH2#1 and ASAH2#2 shown in Table 1 and FIG. 12 are ASAH2obtained from different batches.

TABLE 1 LC/MS^(E) analysis results for L5 without treatment and L5treated with ASAH2 for 24 hours 24 hour baseline ASAH2#1 treatmentASAH2#2 treatment Signal 459.6464 295.3353 202.1154 443.4776 236.9632177.1598 449.8201 230.7273 173.031 451.5772 249.0337 175.7823 Mean451.1303 253.0149 182.0221 Standard 6.658431 29.21906 13.50503 DeviationDecrease 43.9 59.724-hour baseline represents ceramide content of L5 without treatment.

According to Table 1 and FIG. 12, it is known that after L5 was treatedwith ASAH2 for 24 hours, the ceramide content of L5 decreasedsignificantly.

(2) LC/MS^(E) Analysis for L5 Treated with ASAH2 for 24 Hours (for theDetailed Experimental Methods, Please See “5. Efficacy Test for ASAH2”in “A. Method” of Example 1)

L5 was treated with ASAH2 for 24 hours. LC/MS^(E) analysis was performedon L5 without treatment and L5 with the preceding treatment to determinethe ceramide content in the L5 samples mentioned above.

The results for LC/MS^(E) analysis are shown in Table 2 (the four valuesshown in each group were obtained from determining the same sample fourtimes). Conversion was performed to signal of each sample in Table 2 toobtain ceramide content percentage of each sample (the highest signal ofthe L5 without treatment was set as 100%), and the results are shown inFIG. 13. ASAH2#1 and ASAH2#2 shown in Table 2 and FIG. 13 are ASAH2obtained from different batches.

TABLE 2 LC/MS^(E) analysis results for L5 without treatment and L5treated with ASAH2 for 24 hours 24 hour baseline ASAH2#1 treatmentASAH2#2 treatment Signal 2008.465 1827.823 1638.186 2007.321 1747.4221627.067 1946.985 1725.032 1622.848 1989.728 1688.382 1616.651 Mean1988.125 1747.165 1626.188 Standard 28.73594 59.02271 9.070668 DeviationDecrease 12.11997 18.2049524 hour baseline represents ceramide content of L5 without treatment.

According to Table 2 and FIG. 13, it is known that after L5 was treatedwith ASAH2 for 24 hours, the ceramide content of L5 decreasedsignificantly.

(3) LC/MS^(E) Analysis for L5 Treated with ASAH2 in the Presence orAbsence of a Buffer for 2 or 24 Hours

In the presence or absence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 MNaCl, 25 mM CaCl₂), L5 was treated with ASAH2 for 2 or 24 hours.LC/MS^(E) analysis was performed on L5 without treatment and L5 with thepreceding treatment to determine the ceramide content in the L5 samplesmentioned above (for the detailed experimental methods, please see “5.Efficacy test for ASAH2” in “A. Method” of Example 1 except the part ofmixing with the buffer or not).

Conversion was performed to signal of each sample to obtain the ceramidecontent percentage of each sample (the highest signal of the L5 withouttreatment and kept for 2 hours was set as 100%), and the results areshown in FIG. 14. ASAH2#1 and ASAH2#2 shown in FIG. 14 are ASAH2obtained from different batches. In FIG. 14, LDL baseline representsceramide content of L5 without treatment and kept for 0 hour; LDL 2hours represents ceramide content of L5 without treatment and kept for 2hour; LDL 24 hours represents ceramide content of L5 without treatmentand kept for 24 hour.

According to FIG. 14, it is known that, in the presence of a buffer,after L5 was treated with ASAH2 for 2 hours, the ceramide content of L5decreased significantly. Moreover, in the presence or absence of abuffer, after L5 was treated with ASAH2 for 24 hours, the ceramidecontent of L5 both decreased significantly.

(4) LC/MS^(E) Analysis for L5 Treated with ASAH2 in the Presence orAbsence of a Buffer for 24 Hours

In the presence or absence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 MNaCl, 25 mM CaCl₂), L5 was treated with ASAH2 for 24 hours. LC/MS^(E)analysis was performed on L5 without treatment and L5 with the precedingtreatment to determine the ceramide content in the L5 samples mentionedabove (for the detailed experimental methods, please see “5. Efficacytest for ASAH2” in “A. Method” of Example 1 except the part of mixingwith the buffer or not).

The results for LC/MS^(E) analysis are shown in Table 3 (the four valuesshown in each group were obtained from determining the same sample fourtimes). Conversion was performed to signal of each sample in Table 3 toobtain ceramide content percentage of each sample (the highest signal ofthe L5 without treatment was set as 100%), and the results are shown inFIG. 15. ASAH2#1 and ASAH2#2 shown in Table 3 and FIG. 15 are ASAH2obtained from different batches.

TABLE 3 LC/MS^(E) analysis results for L5 without treatment and L5treated with ASAH2 in the presence or absence of a buffer for 24 hours24 hour baseline ASAH2#1 treatment ASAH2#2 treatment Signal 217.36122.40 121.87 220.16 122.08 121.65 214.80 117.11 123.68 215.96 121.81136.51 Mean 217.07 120.85 125.93 Standard 2.31 2.51 7.11 DeviationDecrease 44.33 41.9924 hour baseline represents ceramide content of L5 without treatment.

According to Table 3 and FIG. 15, it is known that in the presence orabsence of a buffer, after L5 was treated with ASAH2 for 24 hours, theceramide content of L5 both decreased significantly.

(5) LC/MS^(E) Analysis for L5 Treated with ASAH2 for 24 Hours

Quantitative analysis for lipid constituents was performed on L5 and L5treated with ASAH2 for 24 hours by mass spectrometry, and the ceramidecontents of the L5 samples mentioned above were compared. The resultsare shown in FIG. 16A.

(6) LC/MS^(E) Analysis for L5 Treated with ASAH2 in the Presence of aBuffer for 2 Hours

In the presence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mMCaCl₂), L5 was treated with ASAH2 for 2 hours. LC/MS^(E) analysis wasperformed on L5 without treatment and L5 with the preceding treatment todetermine the ceramide content in the L5 samples mentioned above (forthe detailed experimental methods, please see “5. Efficacy test forASAH2” in “A. Method” of Example 1 except the part of mixing with thebuffer or not). The results for LC/MSE analysis are shown in Table 4(the four values shown in each group were obtained from determining thesame sample four times). Conversion was performed to signal of eachsample in Table 4 to obtain ceramide content percentage of each sample(the highest signal of the L5 without treatment was set as 100%), andthe results are shown in FIG. 16B.

TABLE 4 LC/MS^(E) analysis results for L5 without treatment and L5treated with ASAH2 in the presence or absence of a buffer for 2 hoursName of sample Signal L5 0 hour 529.0532 L5 0 hour 498.5066 L5 0 hour478.2745 L5 0 hour 432.8346 L5 + ASAH 2 hours 266.8874 L5 + ASAH 2 hours276.2790 L5 + ASAH 2 hour 282.9767 L5 + ASAH 2 hour 283.6284

According to Table 4 and FIG. 16B, it is known that in the presence of abuffer, after L5 was treated with ASAH2 for 2 hours, the ceramidecontent of L5 decreased significantly.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the disclosed embodiments.It is intended that the specification and examples be considered asexemplary only, with the true scope of the disclosure being indicated bythe following claims and their equivalents.

What is claimed is:
 1. A biochemistry reactive material, comprising: a substrate; and an enzyme composition immobilized on the substrate, wherein the enzyme composition is selected from a group consisting of: a first enzyme for eliminating a glycan residue of an electronegative low-density lipoprotein (electronegative LDL); a second enzyme for eliminating ceramide carried by an electronegative low-density lipoprotein; and a combination thereof, wherein the biochemistry reactive material is capable of eliminating electronegative low-density lipoprotein.
 2. The biochemistry reactive material as claimed in claim 1, wherein the substrate comprises silica gel, cellulose, diethylaminoethyl cellulose (DEAE cellulose), chitosan, polystyrene, polysulfone, polyethersulfone, acrylate resin or polysaccharide.
 3. The biochemistry reactive material as claimed in claim 1, wherein the substrate has a particle structure or a hollow-tube structure.
 4. The biochemistry reactive material as claimed in claim 1, wherein the substrate is a cellulose bead.
 5. The biochemistry reactive material as claimed in claim 1, wherein the substrate is a chitosan bead.
 6. The biochemistry reactive material as claimed in claim 1, wherein the substrate is a cellulose hollow fiber, a polysulfone hollow fiber, epoxy acrylic resin or a polyethersulfone hollow fiber.
 7. The biochemistry reactive material as claimed in claim 1, wherein the first enzyme is sialidase or glycosidase.
 8. The biochemistry reactive material as claimed in claim 7, wherein the sialidase is selected from a group consisting of: neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and O-sialidase bioengineered from human genome, one of the foregoing enzymes obtained through gene transformation, expression and purification, and sialidase from a virus or bacterium (alias, acetylneuraminyl hydrolase).
 9. The biochemistry reactive material as claimed in claim 7, wherein the glycosidase is selected from a group consisting of: alpha- and beta-glucosidase bioengineered from human or animal genome, maltase-glucoamylase and sucrase-isomaltase, one of the foregoing enzymes obtained through gene transformation, expression and purification, and N-glycosidase F (PNGase F) and glucosidase from a virus or bacterium.
 10. The biochemistry reactive material as claimed in claim 1, wherein the second enzyme is ceramidase.
 11. The biochemistry reactive material as claimed in claim 10, wherein the ceramidase is selected from a group consisting of: N-acylsphingosine amidohydrolase 1 (ASAH1), N-acylsphingosine amidohydrolase 2 (ASAH2), N-acylsphingosine amidohydrolase 2B (ASAH2B), N-acylsphingosine amidohydrolase 2C (ASAH2C), N-acylethanolamine acid amidase, alkaline ceramidase 1, alkaline ceramidase 2 and alkaline ceramidase
 3. 12. The biochemistry reactive material as claimed in claim 1, wherein the enzyme composition is the first enzyme.
 13. The biochemistry reactive material as claimed in claim 12, wherein the first enzyme is neuraminidase
 2. 14. The biochemistry reactive material as claimed in claim 1, wherein the enzyme composition is the second enzyme.
 15. The biochemistry reactive material as claimed in claim 14, wherein the second enzyme is N-acylsphingosine amidohydrolase
 2. 16. The biochemistry reactive material as claimed in claim 1, wherein the enzyme composition is the combination of the first enzyme and the second enzyme.
 17. The biochemistry reactive material as claimed in claim 16, wherein the first enzyme is neuraminidase 2, and the second enzyme is N-acylsphingosine amidohydrolase
 2. 18. The biochemistry reactive material as claimed in claim 1, wherein the electronegative low-density lipoprotein comprises electronegative low-density lipoprotein L1, L2, L3, L4 or L5.
 19. The biochemistry reactive material as claimed in claim 18, wherein the electronegative low-density lipoprotein is electronegative low-density lipoprotein L5.
 20. A biochemistry reactive device, comprising: the biochemistry reactive material as claimed in claim 1; and a container for containing the biochemistry reactive material, wherein the container has at least one inlet and at least one outlet, wherein a liquid sample enters into the biochemistry reactive device from the at least one inlet, and flows through the biochemistry reactive material to react with the biochemistry reactive material, and then flows out through the at least one outlet.
 21. The biochemistry reactive device as claimed in claim 20, wherein a material of the container comprises glass, acrylic, polypropylene, polyethylene, stainless steel or titanium alloy.
 22. The biochemistry reactive device as claimed in claim 20, further comprising: a filtering material configured in the container behind the at least one inlet and at least one outlet, wherein a pore size of the filtering material is smaller than the biochemistry reactive material to prevent the biochemistry reactive material leaking from the at least one inlet and/or least one outlet.
 23. The biochemistry reactive device as claimed in claim 20, wherein a material of the filtering material comprises filter paper, glass, acrylic, polypropylene or polyethylene.
 24. The biochemistry reactive device as claimed in claim 20, wherein the container is a hollow column, and two ends of the container have a first inlet of the at least one inlet and a first outlet of the at least outlet, respectively.
 25. The biochemistry reactive device as claimed in claim 24, a second inlet of the at least one inlet and a second outlet of the at least outlet are located at a side wall of the hollow column.
 26. The biochemistry reactive device as claimed in claim 20, wherein the substrate has a particle structure or a hollow-tube structure.
 27. The biochemistry reactive device as claimed in claim 22, wherein the substrate has a particle structure or a hollow-tube structure.
 28. The biochemistry reactive device as claimed in claim 22, wherein the substrate has a particle structure.
 29. The biochemistry reactive device as claimed in claim 24, wherein the substrate has a particle structure or a hollow-tube structure.
 30. The biochemistry reactive device as claimed in claim 25, wherein the substrate has a hollow-tube structure.
 31. The biochemistry reactive device as claimed in claim 20, wherein the substrate comprises silica gel, cellulose, diethylaminoethyl cellulose, chitosan, polystyrene, polysulfone, polyethersulfone, acrylate resin or polysaccharide.
 32. The biochemistry reactive device as claimed in claim 28, wherein the substrate is a cellulose bead.
 33. The biochemistry reactive device as claimed in claim 28, wherein the substrate is a chitosan bead.
 34. The biochemistry reactive device as claimed in claim 30, wherein the substrate is a cellulose hollow fiber, a polysulfone hollow fiber, epoxy acrylic resin or a polyethersulfone hollow fiber.
 35. The biochemistry reactive device as claimed in claim 20, wherein the first enzyme is sialidase or glycosidase.
 36. The biochemistry reactive device as claimed in claim 35, wherein the sialidase is selected from a group consisting of: neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and O-sialidase bioengineered from human genome, one of the foregoing enzymes obtained through gene transformation, expression and purification, and sialidase from a virus or bacterium (alias, acetylneuraminyl hydrolase).
 37. The biochemistry reactive device as claimed in claim 35, wherein the glycosidase is selected from a group consisting of: alpha- and beta-glucosidase bioengineered from human or animal genome, maltase-glucoamylase and sucrase-isomaltase, one of the foregoing enzymes obtained through gene transformation, expression and purification, and N-glycosidase F (PNGase F) and glucosidase from a virus or bacterium.
 38. The biochemistry reactive device as claimed in claim 20, wherein the second enzyme is ceramidase.
 39. The biochemistry reactive device as claimed in claim 38, wherein the ceramidase is selected from a group consisting of: N-acylsphingosine amidohydrolase 1, N-acylsphingosine amidohydrolase 2, N-acylsphingosine amidohydrolase 2B, N-acylsphingosine amidohydrolase 2C, N-acylethanolamine acid amidase, alkaline ceramidase 1, alkaline ceramidase 2 and alkaline ceramidase
 3. 40. The biochemistry reactive device as claimed in claim 20, wherein the enzyme composition is the first enzyme, and the first enzyme is neuraminidase
 2. 41. The biochemistry reactive device as claimed in claim 20, wherein the enzyme composition is the second enzyme, and the second enzyme is N-acylsphingosine amidohydrolase
 2. 42. The biochemistry reactive device as claimed in claim 20, wherein the enzyme composition is the combination of the first enzyme and the second enzyme, and the first enzyme is neuraminidase 2 and the second enzyme is N-acylsphingosine amidohydrolase
 2. 43. The biochemistry reactive device as claimed in claim 20, wherein the electronegative low-density lipoprotein comprises electronegative low-density lipoprotein L1, L2, L3, L4 or L5.
 44. The biochemistry reactive device as claimed in claim 20, wherein the electronegative low-density lipoprotein is electronegative low-density lipoprotein L5.
 45. The biochemistry reactive device as claimed in claim 20, wherein the liquid sample comprises aqueous solution, blood or plasma.
 46. A method for ex vivo treating blood or plasma, comprising: (a) ex vivo contacting a blood or plasma with an enzyme composition to react the enzyme composition with the blood or plasma, wherein the enzyme composition is capable of eliminating electronegative low-density lipoprotein, and the enzyme composition is selected from a group consisting of: a first enzyme for eliminating a glycan residue of an electronegative low-density lipoprotein (LDL); a second enzyme for eliminating ceramide carried by a electronegative low-density lipoprotein (LDL); and a combination thereof; and (b) terminating contact between the blood or plasma and the enzyme composition to terminate the reaction of the enzyme composition with the blood or plasma.
 47. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the step (a) is performed for about 0.25-8 hours.
 48. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the step (a) is performed at about 4-40° C.
 49. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the step (a) is performed at about pH 5-10.
 50. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the first enzyme is sialidase or glycosidase.
 51. The method for ex vivo treating blood or plasma as claimed in claim 50, wherein the sialidase is selected from a group consisting of: neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and O-sialidase bioengineered from human genome, one of the foregoing enzymes obtained through gene transformation, expression and purification, and sialidase from a virus or bacterium (alias, acetylneuraminyl hydrolase).
 52. The method for ex vivo treating blood or plasma as claimed in claim 50, wherein the glycosidase is selected from a group consisting of: alpha- and beta-glucosidase bioengineered from human or animal genome, maltase-glucoamylase and sucrase-isomaltase, one of the foregoing enzymes obtained through gene transformation, expression and purification, and N-glycosidase F (PNGase F) and glucosidase from a virus or bacterium.
 53. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the second enzyme is ceramidase.
 54. The method for ex vivo treating blood or plasma as claimed in claim 53, wherein the ceramidase is selected from a group consisting of: N-acylsphingosine amidohydrolase 1, N-acylsphingosine amidohydrolase 2, N-acylsphingosine amidohydrolase 2B, N-acylsphingosine amidohydrolase 2C, N-acylethanolamine acid amidase, alkaline ceramidase 1, alkaline ceramidase 2 and alkaline ceramidase
 3. 55. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the enzyme composition is the first enzyme.
 56. The method for ex vivo treating blood or plasma as claimed in claim 55, wherein the first enzyme is neuraminidase
 2. 57. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the enzyme composition is the second enzyme.
 58. The method for ex vivo treating blood or plasma as claimed in claim 57, wherein the second enzyme is N-acylsphingosine amidohydrolase
 2. 59. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the enzyme composition is the combination of the first enzyme and the second enzyme.
 60. The method for ex vivo treating blood or plasma as claimed in claim 59, wherein the first enzyme is neuraminidase 2, and the second enzyme is N-acylsphingosine amidohydrolase
 2. 61. The method for ex vivo treating blood or plasma as claimed in claim 46, wherein the enzyme composition is immobilized on the substrate.
 62. The method for ex vivo treating blood or plasma as claimed in claim 61, the substrate comprises silica gel, cellulose, diethylaminoethyl cellulose, chitosan, polystyrene, polysulfone, polyethersulfone, resin or polysaccharide.
 63. The method for ex vivo treating blood or plasma as claimed in claim 61, the substrate has a particle structure or a hollow-tube structure. 